U.S. patent number 4,568,649 [Application Number 06/468,558] was granted by the patent office on 1986-02-04 for immediate ligand detection assay.
This patent grant is currently assigned to Immunex Corporation. Invention is credited to Jacques H. Bertoglio-Matte.
United States Patent |
4,568,649 |
Bertoglio-Matte |
February 4, 1986 |
Immediate ligand detection assay
Abstract
A test kit and assay for detecting the presence of minute
amounts of an organic reactant in a test sample includes a
plurality of beads or other types of support structures that are
impregnated with a fluorescer and coated with a ligand that
specifically binds to the organic reactant being investigated. The
beads are placed in an aqueous solution, together with the reactant
which has been radiolabeled. The portion of the radiolabeled
reactant that binds to the ligand is thereby brought in close
enough proximity to the beads to activate the fluorescer to produce
light energy. The radiolabeled reactant that does not bind to the
ligand is, for the most part, disposed too far away from the beads
to enable the radioactive energy emitted thereby to reach the
fluorescer integrated into the beads. Thus, the level of light
energy produced by the fluorescer is indicative of the amount of
reactant present in the test sample.
Inventors: |
Bertoglio-Matte; Jacques H.
(Seattle, WA) |
Assignee: |
Immunex Corporation (Seattle,
WA)
|
Family
ID: |
23860291 |
Appl.
No.: |
06/468,558 |
Filed: |
February 22, 1983 |
Current U.S.
Class: |
436/534; 436/537;
436/800; 436/804; 436/815; 436/817; 436/824 |
Current CPC
Class: |
G01N
33/542 (20130101); G01N 33/585 (20130101); Y10S
436/824 (20130101); Y10S 436/817 (20130101); Y10S
436/804 (20130101); Y10S 436/815 (20130101); Y10S
436/80 (20130101) |
Current International
Class: |
G01N
33/58 (20060101); G01N 33/542 (20060101); G01N
33/536 (20060101); G01N 033/52 (); G01N 033/54 ();
G01N 033/58 () |
Field of
Search: |
;436/508,531,534,537,800,808,824,828 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Virtanen et al., Chemical Abstracts, 93 (1980) #230464d. .
Lehtola et al., Chemical Abstracts, 97 (1982) #203286y. .
Wurzburger et al, J. Pharmacol. Exp. Therapeuti., vol. 203 (1977)
435-41. .
Hart and Greenwald, "Scintillation Proximity Assay (SPA)--A New
Method of Immunoassay", Molecular Immunology, 265 (1979). .
Hart and Greenwald, "Scintillation-Proximity Assay of
Antigen-Antibody Binding Kinetics: Concice Communication", 20,
Journal of Nuclear Medicine, 1062 (1979)..
|
Primary Examiner: Nucker; Christine M.
Attorney, Agent or Firm: Christensen, O'Connor, Johnson
& Kindness
Claims
The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. An immediate ligand detection process, comprising:
(a) placing in an aqueous suspension a plurality of support
particles impregnated with a fluorescer and to which ligands have
been attached;
(b) adding to the suspension fluid a radiolabeled sample reactant
capable of specifically biochemically binding to the ligand, said
radiolabeled sample reactant emitting radiation energy capable of
activating the fluorescer, upon the binding of the sample reactant
to the ligand, the sample reactant is disposed in close enough
proximity to the support particles to cause the radition energy
from the sample reactant to activate the fluorescer to produce
light energy, whereas the sample reactant that does not bind to the
ligand is generally too far removed from the support particles to
enable the radioactive energy to activate the fluorescer; and
(c) measuring the light energy emitted by the fluorescer with the
entire quantities of the support particles and radiolabeled sample
reactant remaining together in aqueous suspension.
2. The process of claim 1, wherein the fluorescer is insoluble in
water.
3. The process of claim 2, wherein the fluorescer is
diphenyloxazole.
4. The process of claim 2, further including impregnating the
support particles with a fluorescer by:
(a) placing the fluorescer in solution in a solvent that is
miscible in water;
(b) adding support particles to the solution, said support
particles being porous to the solution;
(c) removing the support particles from the solution; and
(d) exposing the support particles to water to precipitate the
fluorescer impregnated therein.
5. The process of claim 4, wherein the fluorescer is
diphenyloxazole.
6. The process of claim 5, wherein the solvent is dimethyl
sulfoxide.
7. The process of claim 4 further including the step of dehydrating
the support particles by soaking them in the solvent before adding
them to the solution.
8. The process of claim 1, wherein the sample reactant is labeled
with beta ray or auger electron producing radioactive material.
9. A competitive immediate ligand detection assay process,
comprising:
combining together in an aqueous medium:
a sample to be assayed containing an unknown amount of cold ligand
reactant;
a known quantity of radiolabeled ligand reactant; and
a plurality of support particles having fluorescer integrated
therewith capable of emitting photons when activated by the
radiation energy emitted by the radiolabeled ligand and having
ligand attached to the outer surface thereto, said ligand capable
of indiscriminately binding with both said cold and radiolabeled
ligand reactant whereupon the binding of the ligand with the
radiolabeled ligand reactant positions the radiolabeled ligand
reactant close enough to the support particles to activate the
fluorescer to emit photons through the aqueous medium, whereas the
unbound radiolabeled ligand reactant is generally positioned too
far away from the support particles to enable the radioactive
energy emitted thereby to activate the fluorescer; and
measuring the photons emitted by the fluorescer with the entire
quantities of cold ligand reactant containing sample, radiolabeled
ligand reactant and support particles remaining combined together
in the aqueous medium.
10. The assay of claim 9, wherein the fluorescer is insoluble in
water.
11. The assay of claim 10, wherein the fluorescer includes
diphenyloxazole.
12. The assay of claim 10 further including integrating the
fluorescer with the support particles by:
(a) placing the fluorescer in solution in a solvent that is
miscible in water;
(b) adding the support particles to the solution, said support
particles being porous to the solution;
(c) removing the support particles from the solution; and
(d) exposing the support particles to water to precipitate the
fluorescer integrated with the support particles.
13. The assay of claim 12, wherein the fluorescer includes
diphenyloxazole.
14. The assay of claim 13, wherein the solvent includes dimethyl
sulfoxide.
15. The assay of claim 12, further including the step of
dehydrating the support particles by soaking them in the solvent
before adding the particles to the solution.
Description
TECHNICAL FIELD
The present invention relates to an assay for detecting the
presence of small amounts of organic material, and more
particularly to an assay to detect the presence of organic
materials by monitoring the light energy produced when radioactive
organic molecules of interest biochemically and specifically bind
to a binding structure.
BACKGROUND OF THE INVENTION
In clinical applications, in research and in industry, a need
exists to detect the presence of minute amounts of organic
material, such as antigens, antibodies, hormones, metabolites,
enzymes, and drugs. In clinical situations, detecting the presence
or absence of metabolites, hormones, or other organic factors in
serum or in other body fluids may be useful in the diagnosis of
many clinical conditions, such as pregnancy, infection, blood
disorders, hepatitis, etc. The detection and quantitative analysis
of organic agents is often required in immunological, biological,
chemical, or other types of scientific and medical research. In
industry, assays for organic materials are utilized in quality
control procedures for the production of chemicals and in
monitoring the pollution of water.
Of the numerous chemical and biological assays that have been
developed to detect organic materials, of relevance to the present
invention are precipitation and agglutination assays. In a typical
precipitation assay, the organic material interacts with a reactant
to form a complex that falls out of solution. In agglutination
reactions, the organic substance of interest cross-links an
insoluble reactant to cause the reactant to flocculate. Optical
scattering techniques are commonly used to measure the
flocculation. A drawback of precipitation and agglutination assays
is that they are not as sensitive as radioimmunoassays. Also,
although optical techniques have been developed to improve the
sensitivity of these assays, these techniques require specialized
equipment and analysis.
Another type of known assay for organic materials involves labeling
either the organic material or a reactant thereto with a
radioactive, fluorescent or other type of tracer substance to
ascertain the extent to which the organic material has coupled with
its reactant.
Radioimmunoassay is one of the most common types of these "tracer"
assays. Radioimmunoassay involves combining a known amount of
radiolabeled organic material with a sample containing an unknown
amount of unlabeled organic material of interest together with a
specific antibody that binds indiscriminantly to the labeled and
unlabeled organic materials to form a complex. After an incubation
period, the unbound organic materials are separated from the bound
organic materials, typically by precipitation of the complex with
polyethylene glycol, adsorption of the unbound material with
activated charcoal or utilization of solid-phase reagents. Then the
radioactivity of either of these two fractions is measured. A
certain amount of the labeled and unlabeled organic material will
be bound to the reactant, with the amount of the bound labeled
organic material being inversely related to the quantity of
unlabeled inorganic material present in the sample being
tested.
A drawback of the radioimmunoassay is that the procedures for
separating the bound organic materials from the unbound require a
significant number of time-consuming operations that are often
complicated and expensive. The separation procedures involve
repeatedly washing the complex of organic material and antibody
with a rinsing solution and/or centrifuging the mixture to remove
the unbound organic material from the reactant, thereby generating
radioactive waste material with each washing. By the time that the
separation process has been completed, significant volumes of
radioactive waste material are produced. This waste material is not
only expensive to dispose of, but also presents a potential health
hazard to persons handling the material, including during the
separation procedures.
In an assay utilizing fluorescence, the organic material may be
labeled with an appropriate fluorescer, such as fluorescein
isothiocyanate. The extent to which the fluorescer labeled organic
materials bound to a specific reactant can be examined under a
light microscope with a suitable light source and filters to
provide incident light of the proper wavelength to cause
fluorescence.
An example of a particular fluorescence technique is disclosed in
U.S. Pat. No. 4,161,515 wherein an unknown organic compound, whose
presence is being investigated, is mixed with: (1) a known quantity
of antibody against the organic compound; (2) an organic analog,
having a fluorescer bound thereto, which competes with the unknown
organic compound for the antibody; and (3) an antibody for the
fluorescer. The competition between the unknown organic material
and the known analog-fluorescer effectively reduces the
concentration of the antibody, thereby causing more of the
fluorescer-antibody to combine with the analog-fluorescer. This, in
turn, causes a corresponding change in the emission spectrum of the
fluorescer.
A tracer assay that utilizes both fluorescence and radioactive
substances is disclosed by U.S. Pat. No. 4,000,252 wherein a known
quantity of radiolabeled antigen and a sample containing an unknown
amount of unlabeled antigen are placed within an
immunoscintillation cell. An insolubilized or solid phosphor, which
is chemically or physically associated with an antibody to the
antigens, is also added to the cell. The unbound antigens are
washed from the cell and then the luminescence emitted by the
phosphor due to activation from the radioactive energy from the
found labeled antigens is measured inside a scintillation counter.
Removal of the unbound antibodies from the cell requires several
washing procedures that are not only time consuming, but also
produce significant quantities of radioactive waste material.
Assays that combine tracer techniques with agglutination are
disclosed by U.S. Pat. Nos. 4,018,972 and 4,271,139. In U.S. Pat.
No. 4,108,972, microscopic carrier particles, each containing a
fluorescent tracer material, and a biological reactant to the
antibody or antigen being investigated, are placed in suspension.
When the antigen or antibody is added to the suspension, it binds
to the reactant coating to cause flocculation of the carrier
particles. The flocculated material is then separated from the
suspension fluid and other constituents by numerous washings.
Thereafter, the flocculated material is dissolved and then assayed
by flourescence techniques to determine the quantity of organic
material present.
In U.S. Pat. No. 4,271,139, tritiated (radioactive) latex particles
coated with antigen and polystyrene scintillant particles coated
with the same antigen were placed in an aqueous medium with a
sample containing an unknown quantity of a corresponding antibody.
The number of tritiated antigen coated latex particles linked to
the antigen coated scintillant particles is related to the
concentration of antibody present. Also, when the two particles are
linked together by the antibody, the radioactive energy from the
tritiated particles initiates scintillation within the scintillant
particles. Scintillations are then measured by an appropriate
detector, with the detected level of scintillation being indicative
of the quantity of antibody present. Addition of an unknown
quantity of non-radioactive antigen then competes with the antigen
coated bead binding to the antibody, thereby reducing bead
agglutination, scintillation and signal. A drawback of this
particular assay is that it requires both tritiated latex particles
and polystyrene scintillant particles to be coated with antigen,
which increases the expense and complexity of the assay. In
addition, relative to the standard radioimmunoassay discussed
above, extremely large suspension volumes are required for the
assay to operate properly. The assay process of the '139 patent
also requires the availability of relatively pure antigen to be
used to bind to the two types of carrier particles. Relatively pure
samples of antigen are both expensive and difficult to obtain.
Thus, it is a principal object of the present invention to provide
an accurate, inexpensive, and rapid assay procedure and test kit
for detecting the presence of extremely small amounts of organic
materials.
A particular object of the present invention is to provide an assay
of equivalent accuracy to present techniques, but which does not
require highly skilled personnel or large amounts of time to
perform using standard commercially available equipment.
A further particular, but highly important object of the present
invention, is to provide an assay procedure that produces only a
minimum volume of radioactive waste and requires only a minimum
amount of handling of hazardous substances.
An additional particular object of the present invention is to
provide an assay that can be used to rapidly test a large number of
samples.
Another particular object of the present invention is to provide an
assay that utilizes water as a suspension medium.
SUMMARY OF THE INVENTION
The foregoing and other objects are achieved in accordance with the
present invention by providing a test kit and assay procedure which
produces light energy at a level related to the amount of organic
material present in a sample being tested. The light energy is
produced by a fluorescer which is integrated into support bodies in
the form of beads or other structures. The support bodies are
coated with a ligand that is capable of specifically binding to the
organic material or reactant of interest. When the present
invention is used as a direct assay, the reactant is radiolabeled.
Then, the sample containing the radiolabeled reactant is mixed in
an aqueous solution with the support bodies, causing the reactant
to bind to the ligand. This places the radiolabeled reactant in
close enough physical proximity to the support bodies to cause the
radiation energy emitted from the radiolabeled reactant to activate
the fluorescer integrated into the support bodies, thereby causing
the fluorescer to emit light energy. The level of the light energy
produced is related to the amount of reactant that is bound to the
ligand, which in turn, is indicative of the amount of reactant
present in the sample being tested.
The present invention may also be utilized as a competitive assay
to determine the amount of a reactant contained in a sample. In
this situation, a known amount of the reactant is radiolabeled. The
reactant of interest in the sample being tested remains unlabeled.
Both the labeled and unlabeled reactant are capable of specifically
binding with the ligand. In the assay process, both the labeled and
unlabeled organic materials are placed in an aqueous solution,
together with the support bodies that have been impregnated with
the fluorescer and coated with the ligand. Since the ligand does
not favor either the labeled or unlabeled reactant, the reactants
bind to the ligand in proportion to their relative amounts present
in the aqueous solution. However, only the radiolabeled reactant
that binds to the ligand is brought in close enough proximity to
the support bodies to cause the radiation energy emitted thereby to
activate the fluorescer integrated into the support bodies. Thus,
the level of light energy produced by the fluorescer is inversely
proportional to the quantity of unlabeled reactant present in the
sample.
In both the direct and competitive assays, radiation energy emitted
by the radiolabeled reactant, which is capable of activating the
fluorescer, has a limited range of travel in water. Thus, the
reactant that has not bound to the ligand is too far away from the
support structures to permit the radiation energy emitted therefrom
to reach the fluorescer. Thus, since only the radiolabeled reactant
that actually binds to the ligand is responsible for causing the
fluorescer to emit light energy, the radioactive reactant that has
not bound to the ligand need not be separated from the
ligand-reactant complex prior to measuring the level of light
energy emitted by the fluorescer. Thereby, as a consequence of the
present invention, the laborious and time-consuming procedure of
separating the unbound labeled reactant from the bound complexes by
centrifuge, precipitation, washing and other procedures is
eliminated, as are the large quantities of radioactive waste
material produced by these separation techniques.
In a further aspect of the present invention, a unique technique is
provided for integrating the fluorescer into the support bodies.
Initially, the support bodies are soaked in a solvent for the
fluorescer which is miscible in water to dehydrate the bodies.
Thereafter, the bodies are placed in a solution composed of the
fluorescer and solvent so that the fluorescer is integrated and/or
adsorbed into the bodies. Then, the bodies are removed from the
solvent and then placed in an aqueous solution which causes
precipitation of the fluorescer within the bodies, thereby locking
the fluorescer therein. By this technique, the fluorescer is
integrated within the interior of the bodies so that the
radiolabeled reactant is placed in very close proximity to the
fluorescer upon binding to the ligand, which is disposed on the
exterior of the bodies.
DETAILED DESCRIPTION
In accordance with the present invention, support bodies or
particles in the form of beads or other structures are impregnated
and/or coated with a material capable of fluorescence when excited
by radioactive energy. The beads are coated with a ligand that is
capable of specifically binding to a reactant of interest by
covalently linking or directly attaching the ligand to the beads.
The beads are then mixed in a water-based solution containing the
reactant that has been radiolabeled. Upon binding of the
radiolabeled reactant to the ligand, the fluorescer integrated into
the beads is placed in close enough physical relationship to the
reactant to allow the radiation energy emitted from the reactant to
activate the fluorescer thereby causing the fluorescer to emit
light energy. The level of light energy emitted, which is
indicative of the extent to which the ligand is bound to its
reactant, may be conveniently measured with a scintillation counter
or other monitoring device employing a photomultiplier tube.
The radiation energy emitted by the radiolabeled reactant molecules
has a very limited range of travel in water. The reactant molecules
that have not bound to the ligand are, for the most part, located
too far away from the ligand to enable the radiation energy emitted
from these unbound reactant molecules to reach the fluorescer in
the support structure, i.e., beads. Since there is very little
likelihood of chance excitation of the fluorescer by the
radioactive energy of these unbound reactant molecules, the
reactant molecules that have not bound to the ligand need not be
separated from the ligand-reactant complexes prior to scintillation
counting of the light energy emitted by the fluorescer excited by
radioactive energy from the reactant molecules that have bound to
the ligand. Thus, the traditional laborious and potentially
hazardous procedure of separating the unbound reactant from the
ligand-reactant complexes is eliminated.
The present invention may also be used in conjunction with a
competitive assay procedure. In this instance, the support bodies,
having a fluorescer integrated therein and coated with an
appropriate ligand, are placed in an aqueous solution containing a
known quantity of radiolabeled reactant and a sample containing an
unknown amount of the same, but unlabeled reactant. Since the
ligand does not favor binding to either the labeled or unlabeled
reactant over the other, the amount of labeled reactant binding to
the ligand will be inversely proportional to the quantity of
unlabeled reactant present in the sample. Prior to the assaying of
a particular sample, different known amounts of unlabeled reactant
are mixed together in individual vials with constant amounts of
radiolabeled reactant and with a fixed quantity of ligand coated
beads. The level of fluorescent energy generated by excitation of
the fluorescer from the radiolabeled reactant that has bound to the
ligand is measured for each vial containing a known amount of the
unlabeled reactant. From the results of these measurements, a
standard curve may be prepared depicting the level of fluorescent
energy measured per quantity of unlabeled reactant present. Then,
when a particular sample containing an unknown amount of unlabeled
reactant is assayed, the concentration of the unlabeled reactant in
the sample may be determined from the standard curve once the level
of fluorescent energy being emitted is measured.
As in the direct assay procedure described above, in the
competitive assay, only the radiolabeled reactant molecules that
are bound to the ligand are in close enough proximity to the beads
to allow the radiation energy emitted by the labeled reactant to
bombard the fluorescer integrated into the beads. The detected
level of fluorescent energy, therefore, is a reflection of the
proportion of the radiolabeled reactant which actually binds to the
ligand. As a consequence, there is no need to wash the beads or
otherwise attempt to remove the unbound radioactive reactant from
the ligand; instead, the level of fluorescent energy may be
measured with all of the components of the assay still present in
the vial. Moreover, the time required to complete the assay is
limited only by the ligand-reactant binding reaction rate of the
system under investigation.
As noted above, the ligand is bonded to and the fluorescer is
integrated with a structural support, such as beads. Various types
of beads may be utilized, such as polyacrylamide, acrylamide,
agarose, polystyrene, polypropylene, polycarbonate or Sepharose 4B
beads (from Pharmacia Fine Chemicals, Uppsala, Sweden). The present
invention also may be carried out with other shapes or types of
support structures, for instance latex particles, as long as the
ligand molecules can be covalently or otherwise attached thereto
and a fluorescer integrated therewith.
Beads, such as the Sepharose 4B beads noted above, are commercially
available in an activated state. Compounds, such as cyanogen
bromide, are incorporated in the beads to covalently bind with
certain ligands. The process by which the ligand is bound to the
beads is dependent on the type of bead and the particular ligand
employed. For instance, for Sepharose 4B beads activated with
cyanogen-bromide, a ligand in the form of Staphylococcus aureus
protein A or an antibody to a specific reactant may be bound to the
beads by placing the beads in a solution containing the protein A
or antibody and an appropriate buffer. Thereafter, the excess
protein A or antibody is washed away and the remaining active sites
on the beads to which no protein A or antibody had attached are
blocked with an appropriate blocking agent, such as glycine. This
prevents the reactant of interest and others from binding directly
to the beads, rather than to the ligand. Other techniques for
bonding a ligand to beads include the use of carbodiimide coupler,
tannic acid, glutaraldehyde and polyethylene glycol.
As noted above, in accordance with the present invention, a
fluorescer is integrated within the beads to give off light energy
when radiolabeled reactant is brought in close enough proximity to
the fluorescer to cause excitation thereof, i.e., by binding to the
ligand on the bead surface. Various types of fluorescers may be
used; however, since the process of the present invention takes
place in an aqueous solution, the fluorescer must be insoluble in
water so that it does not dissociate from the beads during the
assay procedure. Also, the fluorescer employed must be excitable to
a higher energy state by the particular wavelength of the
radioactive energy rays emitted by the radiolabeled reactant, and
also must release sufficient light energy when returning to its
normal energy state to be detected by a scintillation counter or
other detection device utilizing a photomultiplier tube. An example
of a fluorescer that has been found to meet these requirements for
use with radioactive energy in the form of beta rays or auger
electrons is diphenyloxazole (hereinafter "PPO").
The present invention involves a novel process for integrating a
fluorescer into a support structure, such as beads. Since the
fluorescer is insoluble in water, an appropriate transfer medium,
in which the fluorescer is soluble, must be used to incorporate the
fluorescer into the beads. Moreover, the transfer medium itself
must be miscible with water.
The novel method of the present invention for integrating the
fluorescer into the beads includes soaking the beads in an
appropriate transfer solvent for the fluorescer that is miscible
with water thereby to dehydrate the beads. Thereafter, the beads
are incubated in a solution of the fluorescer and solvent. The
fluorescer, which is in solution, is absorbed into the bead. The
excess solvent is then discarded and the fluorescer is
precipitated, adsorbed and/or integrated inside the bead by adding
water or a buffered saline solution. Precipitating the fluorescer
within the beads locks the fluorescer therein. Next, the beads are
washed to remove the excess precipitated fluorescer and then
resuspended in a solution containing both a detergent, such as
Tween 20, to prevent the beads from sticking together and gelatin
to bind to any sites on the surface of the beads that either have
not been blocked by the previously employed blocking solution or
bound to a ligand. This lowers the nonspecific binding tendency of
the beads. Finally, a bactericide, such as sodium azide, can be
applied to the beads to prevent the growth of bacteria thereon.
Applicant has found the dimethyl sulfoxide (hereinafter "DMSO") may
be utilized as a transferring solvent if PPO is used as a fluor.
PPO is soluble in DMSO, and DMSO is miscible in water. Also, the
DMSO does not hinder the ability of the PPO to precipitate within
the beads when subjected to an aqueous solution.
The process of the present invention requires the use of
radio-labeled reactants. The radiolabeled reactants are
biologically and chemically identical to an unlabeled reactant,
with the exception that the labeled reactants emit radioactive
energy due to the decaying of the radioactive isotope present.
The technique used for labeling of the reactant varies with the
type of radioactive isotope employed. For instance, labeling can be
accomplished by replacing one of the atoms of the reactant
molecules with a corresponding radioactive isotope. A hydrogen atom
could be replaced with: tritium, .sup.3 H; a carbon atom replaced
with carbon-14, .sup.14 C; or a strontium atom replaced with
strontium-38, .sup.38 Sr. In another labeling process, rather than
replacing the atoms of the reactant with a radioactive isotope, an
isotope may be added to the reactant molecule. Such radioactive
isotopes in common use include: iodine-125, .sup.125 I; and
iron-59, .sup.59 Fe. In situations in which biological organisms or
parts of those organisms are capable of synthesizing proteins,
labeling can be carried out by culturing the organism with an
appropriate radiolabeled precurser, such as methionine-35 (.sup.35
S), to cause the organism to incorporate the isotope into its
products. Many reactants, such as antigens, antibodies, hormones,
hormone receptors, enzymes, or enzyme cofactors, are readily
available in radiolabeled form from various commercial sources.
Radioactive isotopes used to label the reactant have only a limited
range in water so that, for the most part, only the radioactive
energy from the labeled reactant that binds to the ligand actually
activates the fluorescer. If PPO is used as a fluorescer, applicant
has found that isotopes that emit either beta rays or auger
electrons from gamma ray emissions fulfill this requirement. PPO
does not fluoresce from the gamma rays themselves which have a
longer range of travel than beta rays or auger electrons.
The assay process of the present invention may be utilized in
conjunction with any ligand-reactant combination or system that
specifically binds together and in which the reactant may be
radiolabeled without affecting its specificity for the ligand.
Examples of such ligand-reactant combinations include antibodies
and their corresponding antigens. Either the antibody or antigen
may be attached to the bead or other type of support structure to
function as the ligand, with the corresponding antigen or antibody
serving as the reactant. Another ligand-reactant system may be
composed of protein A and corresponding immunoglobulins. The need
to ascertain the presence of antigens, antibodies, and
immunoglobulins exists in many clinical and research settings,
especially in the detection of diseases and allergies and in
investigations of the immune system.
Additional ligand-reactant systems with which the present invention
is especially useful include: (1) lectins-glycoproteins; (2)
biotin-avidin; (3) hormone receptor-hormone; (4) enzyme-substrate
or cofactor; (5) RNA-DNA; and (6) DNA-DNA. It is to be understood
that in the present invention either element may serve as the
ligand or reactant.
The present invention also may be of particular value in conducting
enzyme kinetic strudies. The enzyme may serve as a ligand to bind
with the radiolabeled reactant. Since all of the reagents of the
assay system are always present together in the same vial and
because the reagents are suspended in an aqueous-based buffer
rather than an organic solvent, kinetic experiments may be
conveniently carried out by simply measuring the light energy
emitted from the same vial at different time intervals to determine
the reaction rate of the reagents. Also, in the RNA-DNA system, the
commonly used Northern, Southern and Western Blot tests may be
advantageously replaced with the assay of the present
invention.
EXAMPLE I
Immunoglobulin-G Direct Detection Assay With Tritium Labeling
To use the assay of the present invention to detect the presence of
immunoglobulin-G, cyanogen-bromide activated Sepharose 4B beads
(obtained from Pharmacia Fine Chemicals, Uppsala, Sweden) are
covalently coated with Staphyloccus aureus cowan strain 1 protein A
(Pharmacia Fine Chemicals, Uppsala, Sweden) or with
anti-immunoglobulin-G antibody. This is accomplished by swelling
and washing one gram of cyanogen-bromide activated Sepharose 4B
beads in one millimolar hydrochloric acid. Then, two milliliters of
the washed beads are placed in a solution composed of either two
milligrams of protein A or 15 milligrams of the immunoglobulin-G
fraction of a rabbit antiserum to the human immunoglobulin-G heavy
chain, together with sodium bicarbonate buffer, pH 8.3, containing
0.5 molar saline. The suspension is incubated for two hours at room
temperature and then the excess protein is washed away by
centrifuging. Thereafter, the remaining active sites on the beads
are blocked with 0.2 molar glycine. The beads are next washed with
acetate buffer and bicarbonate buffer.
A fluorescer in the form of PPO is next incorporated into the
beads. This is accomplished by dehydrating the coated beads by
soaking them in DMSO for 15 minutes to remove any water in the
beads that would cause premature precipitation of the PPO since PPO
is insoluble in water. This step is repeated twice more and then
the excess solvent removed by sedimentation. The beads are next
incubated for 30 minutes at room temperature in a 20 to 40 percent
weight by volume solution of PPO in DMSO. After incubation, the
excess solution is discarded and the PPO precipitated inside the
beads by adding ten volumes of either phosphate buffered saline
(hereinafter "PBS") or water. The suspension is then washed five
times in PBS to remove the excess precipitated PPO which is not
bound inside the beads. Thereafter, the beads are resuspended at a
final concentration of ten percent volume/volume in PBS
supplemented with 0.5 percent volume/volume Tween 20 as a detergent
to help prevent the beads from sticking together and 0.1 percent
weight/volume gelatin to bind to the remaining sites on the beads
which were not blocked by reaction with protein A, immunoglobulin-G
antibody or the glycine. This minimizes any nonspecific binding of
radiolabeled reactant to the beads. Lastly, sodium azide, NaN.sub.3
in an amount of 0.01 percent weight/volume, can be added to prevent
bacterial growth.
In a direct assay for immunoglobulin-G, 50 microliters of the
protein A or anti-immunoglobulin-G antibody coated beads are mixed
together in scintillation vials with various concentrations of
immunoglobulin-G labeled to a specific activity of
5.times.10.sup.12 counts per minute per millimole with .sup.3
H-acetic anhydride. PBS supplemented with 0.5 percent volume/volume
Tween is added to the vials to bring the total volume in each vial
up to a total of 3.0 milliliters. Then, the light energy produced
by the bombardment of the PPO with the beta rays from the
radiolabeled immunoglobulin-G is directly measured by a
scintillation counter. The results of the assay wherein beads
coated with protein A were utilized, shown in Table I, indicate
that increasingly higher counts per minute were obtained by
increasing the concentration of tritium-labeled immunoglobulin-G in
the sample. PPO-impregnated Sepharose 4B beads that were not coated
with either a protein or anti-immunoglobulin-G antibody were used
as a control, as also shown in Table I.
TABLE I ______________________________________ DIRECT DETECTION
ASSAY FOR HUMAN IMMUNOGLOBULIN-G Tritium Labeled Human Counts Per
Minute Immunoglobulin-G Determined After Bead Source (Micrograms) 5
Minute Incubation ______________________________________ 50
Microliters of 5 4221 PPO Integrated 10 5375 Sepharose 4B 20 6140
Beads Coated 40 7286 with protein A 50 Microliters of 40 35 PPO
Integrated uncoated Sepharose 4B Beads
______________________________________
EXAMPLE II
Immunoglobulin-G Direct Detection Assay With Iodine 125
This Example is identical with Example I, except that the protein A
or anti-immunoglobulin-G antibody coated beads were mixed with
various concentrations of human immunoglobulin-G labeled with
.sup.125 I to a specific activity of 8.times.10.sup.4 counts per
minute per millimolar by the Chloramine T method. The protein A or
anti-immunoglobulin-G antibody coated beads were placed in
scintillation vials with various concentrations of .sup.125 I
labeled human immunoglobulin-G and then the vials were placed
directly into a scintillation counter to measure the resulting
level of photon emission. The results of these tests are shown in
Table II for 5-minute and 60-minute incubation periods. These data
confirm that the assay can be measured immediately after mixing
labeled reactant with PPO-impregnated ligand bound beads.
TABLE II ______________________________________ DIRECT DETECTION
ASSAY FOR HUMAN IMMUNOGLOBULIN-G Counts Per Minute Iodine-125
Determined After Labeled Human 5 min/60 min Bead Source
Immunoglobulin-G Incubations ______________________________________
75 Microliters of PPO 125 ng 10596/10238 Integrated Sepharose 375
ng 14880/14910 4B Beads Coated with 500 ng 18132/19638 protein A
750 ng 26216/27640 1000 ng 33876/37784 1500 ng 40066/41846 75
Microliters of PPO 125 ng 11030/11874 Integrated Sepharose 375 ng
16762/18198 4B Beads Coated with 500 ng 22684/24985 anti-immuno-
750 ng 24088/25946 globulin-G antibody 1000 ng 25904/31514 1500 ng
30056/37068 ______________________________________
EXAMPLE III
Competitive Assay for Immunoglobulin-G
Cyanogen-bromide activated Sepharose 4B beads (Pharmacia Fine
Chemicals, Uppsala, Sweden) are coated with either protein A (0.1
milligrams per milliliter of gel) or anti-immunoglobulin-G antibody
(1.5 milligrams per milliliter of gel) in the manner set forth in
Example I. Also, the Sepharose 4B beads are impregnated with the
fluorescer PPO in the manner discussed in Example I.
Fifty-microliter quantities of the prepared beads are mixed
together in scintillation vials with 0.5 micrograms of .sup.125
I-human immunoglobulin-G and increasing concentrations of unlabeled
human immunoglobulin-G. The samples are then brought up to three
milliliters each by adding PBS supplemented with 0.5 percent
volume/volume of Tween. The sample is then immediately counted for
photon emission levels.
From the photon emission level measurements, standard inhibition
curves were plotted in terms of the percent of inhibition of
binding of the radiolabeled immunoglobulin-G caused by different
quantities of unlabeled immunoglobulin-G. The inhibition curves
using both anti-immunoglobulin-G antibody and protein A as ligands
shown in FIG. 1. A sample containing an unknown amount of
immunoglobulin-G can now be measured for percent of inhibition of
the radiolabeled reactant and from this measurement the quantity of
immunoglobulin-G present can be determined by using FIG. 1.
EXAMPLE IV
Thyroxin Determination
This Example involves the use of the present invention to develop a
standard inhibition curve for the detection of thyroxin.
Cyanogen-bromide activated Sepharose 4B beads were coated with
protein A and then impregnated with PPO in the manner described in
Example I. Two hundred microliters of the prepared beads were
pipetted into individual scintillation vials together with 400
microliters of a rabbit anti-thyroxin antiserum (obtained from
Abbott Laboratories, Chicago, IL). Twenty-five microliters of
various concentrations of unlabeled thyroxin (obtained from Abbott
Laboratories, Chicago, IL) were added to each vial. Next, 100
microliters of thyroxin labeled with the isotope iodine-125
(obtained from Abbott Laboratories, Chicago, IL) were added to the
vials. Lastly, the volume of the reaction mixture in each vial was
brought up to three milliliters by the addition of PBS supplemented
with 0.5 percent volume/volume of Tween. The photon emission level
in each vial was then measured in a scintillation counter and the
results plotted in FIG. 2 on a probability-log format. As shown in
FIG. 2, as the volume of unlabeled thyroxin added to the vials
increased, the proportion of labeled thyroxin that bound to the
beads decreased as expected. This standard inhibition curve can be
used to determine the amount of thyroxin present in an unknown
sample by using the same protocol described above in Example III to
determine the percent of radiolabeled thyroxin that is inhibited
from binding on the beads. From this value, the amount of thyroxin
present in the sample may be conveniently read from the curve.
As will be apparent to those skilled in the art to which the
invention is addressed, the present invention may be embodied in
forms other than those specifically disclosed above without
departing from the spirit or essential characteristics of the
invention. The particular embodiments of the immediate ligand
detection assay method, described above, are therefore to be
considered in all respects as illustrative and not restrictive. The
scope of the present invention is as set forth in the appended
claims rather than being limited to the examples of the immediate
ligand detection method set forth in the foregoing description.
* * * * *